Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture
Michael Lattke1, Robert Goldstone2, James Ellis3, Stefan Boeing4, Jeronimo Jurado-Arjona5, Nicolás Marichal5, James MacRae3, Benedikt Berninger5,6, Francois Guillemot1*
1Neural Stem Cell Biology Laboratory, The Francis Crick Institute, London, NW1 1AT, UK 2Advanced Sequencing Facility, The Francis Crick Institute, London, NW1 1AT, UK 3Metabolomics Facility, The Francis Crick Institute, London, NW1 1AT, UK 4Bionformatics & Biostatistics, The Francis Crick Institute, London, NW1 1AT, UK 5Institute of Psychiatry, Psychology & Neuroscience, Centre for Developmental Neurobiology, King's College London, London, SE1 1UL, UK 6MRC Centre for Neurodevelopmental Disorders, King's College London, London, SE1 1UL, UK
*lead author
Corresponding author e-mail address: Francois.Guillemot@crick.ac.uk
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Summary
Astrocytes have essential functions in brain homeostasis that are established late in differentiation, during astrocyte maturation, but the underlying mechanisms are not well understood. Here we identified extensive transcriptional changes that occur during murine astrocyte maturation and are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lacked expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induced distinct sets of mature astrocytes-specific transcripts. Culturing astrocytes with FGF2 in a three-dimensional gel induced expression of Rorb, Dbx2 and Lhx2 and improved their maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation. This comprehensive analysis is available as online resource (http/…).
Single-cell RNA-Seq analysis of striatal astrocyte maturation (related to Figure 1)
Rationale Astrocytes have essential functions during brain development and homeostasis. Immature astrocytes in the developing brain support developmental processes such as neuronal maturation, while mature astrocytes in the adult brain support brain homeostasis and modulate neuronal signalling. However, the stages of astrocyte development and the molecular mechanisms underlying these functional changes are not well understood. Therefore, to further elcuidate the stages and molecular changes, we have analysed the gene expression changes during astrocyte maturation in the murine striatum, which occurs between birth and around 4 weeks of age, using scRNA-Seq.
Experimental design Astrocytes were acutely purified from striatum tissue at postnatal day 3 (P3, for immature astrocytes) or at 3 months of age (3m, for mature astrocytes) using ACSA2-MACS. Astrocytes from 3 independent preparations (each 2-5 animals) were analysed using 10X-Genomics scRNA-Seq.
Analysis outline Cells with >500 features (detected genes) and < 10% mitochondrial content were clustered using Seurat3 (Figure 1C). Sox9-postive astroglial lineage clusters were isolated and reclustered (Figure 1D). Pseudotime analysis was performed using Slingshot (Figure 1E). Changes in gene expression along pseudotime were identified using tradeSeq, and the identified genes grouped according to their mean expression in pseudotime bins (Figure 1F).
Results summary In the Seurat analysis we identified several clusters of transcriptionally clearly distinct immature and mature astrocytes. Slingshot pseudotime analysis identified a likely maturation trajectory which includes two populations of proliferating progenitors, two consecutive stages of postmitotic immature astrocytes, and two main populations of mature adult astrocytes. Our pseudotemporal gene expression analysis revealed 4 groups of immature astrocyte genes and two groups of mature astrocyte genes with distinct expression patterns.
UMAP clustering, complete dataset (Figure 1C)
Astroglial lineage (Sox9+ cells) reclustered (UMAP, Figure 1D)
Astroglial lineage Slingshot pseudotime analysis (Figure 1E)
Cluster labels from the Seurat reclustering (Figure 1D) were used to infer lineage relationships using Slingshot (using Cluster 6 as starting point (Proliferation-marker expressing progenitors with highest negative PC1 loading)). Lineage 6, which includes the main adult astrocyte clusters 7 and 14 (highest positive PC1 loading), was considered as the main striatal astrocyte differentiation/maturation trajectory.
Gene expression changes along pseudotime (main striatal astrocyte lineage, Figure 1F)
Mean gene expression of all cells in each pseudotime bin (equal pseudotime ranges), centered on the mean of all bins. Bins 1 and 20 were omitted as they contained less than 10 cells each. Genes shown were identified as dynamically regulated by the tradeSeq algorithm. Genes were further filtered to remove lowly changing genes with less than 0.2 deviation from the mean in all bins.
Bulk RNA-Seq analysis of cortical astrocyte maturation (related to Figure 2)
Rationale To validate our the scRNA-Seq analysis of striatal astrocyte maturation and identify transcriptional changes that are common during astrocyte maturation in different brain regions, we analysed maturation of cortical Astrocytes in vivo using bulk RNA-Seq
Experimental design Astrocytes were acutely purified from cortex tissue at postnatal day 4 (P4, for immature astrocytes) or at 2 months of age (2m, for mature astrocytes) using ACSA2-MACS. Astrocytes from 3 independent preparations (each 2-5 animals) were analysed using bulk RNA-Seq.
Analysis outline DESEq2 was used to indentify differentially expressed genes.
Results summary Our analysis identified a high confidence signature of immature and mature astrocyte-specific genes which are changing during maturation in both the striatum and cortex, independent of the different analysis approaches.
Volcano plot showing gene expression changes from P4 to 2m (Figure 2B)
Maturation-regulated genes from striatal the scRNA-Seq analysis are highlighted.
ATAC-Seq analysis of cortical astrocyte maturation to assess changes in chromatin accessibility linked to changes in maturation-regulated genes (Figure 3)
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Expression of transcription factors that might bind motifs enriched in chromatin changing accessibility (Figure 3G)
Immature ETS transcription factors
Mature ROR/HOX transcription factors
Bulk RNA-Seq analysis of astrocyte maturation in vivo vs in vitro (differentiation with BMP4)
Rationale To characterise an in vitro model for the further analysis of the mechanisms underlying astrocyte maturation and to assess to which extent astrocyte maturation in vivo is recapitulated in vitro, we performed RNA-Seq of astrocytes differentiated from cultured neural stem/progenitor cells using BMP4 and compared these to immature and mature cortical astrocytes in vivo (from Figure 2)
Experimental design Astrocyte maturation in vitro was analysed by RNA-Seq of cultured neural stem/progenitor cells and astrocytes differentiated from these cells by growth factor withdrawal and exposure to BMP4 containing medium for 14 days.
Analysis outline DESEq2 was used to indentify differentially expressed genes. Gene expression was compared to immature and mature cortical astrocyte profiles from above, focussing on the common immature/mature astrocyte-enriched genes from the cortical and striatal in vivo analyses (see above, Figures 1/2).
Results summary We show here that astrocytes in our in vitro model fail to induce a large part of the mature astrocyte gene signature, suggesting that they remain partially immature. Among these genes are several transcription factors, whose lack of expression might explain the lack of maturation in vitro.
Selected mature genes in vivo vs in vitro (Figure 4C)
Selected mature transcription factors in vivo vs in vitro (Figure 4G)
Gene expression changes induced by transcription factor expression in vitro (Figure 5C)
Rationale To investigate the role of individual transcription factors in astrocyte maturation, we focussed on four candidate maturation regulators Rorb, Dbx2, Lhx2 and Fezf2, which were not induced during astrocyte differentiation in our in vitro model. In a reconstitution assay, we forced expression of these factors in this model and analysed the consequences for the expression of mature astrocyte genes by RNA-Seq.
Experimental design Astrocyte were differentiated from culutred neural stem cells by growth factor withdrawal and exposure to BMP4 containing medium. At day 6 of differentiation, astrocytes were infected with a lentiviral vector to induce expression of the candidate regulators Rorb, Dbx2, Lhx2, Fezf2 or an EGFP control transgene. RNA-Seq was performed at day 14.
Analysis outline DESEq2 was used to indentify differentially expressed genes. Gene expression was compared to immature and mature cortical astrocyte profiles from above, focussing on the common immature/mature astrocyte-enriched genes from the cortical and striatal in vivo analyses (see above, Figures 1/2).
Results summary We could show that the expression of Rorb, Dbx2, Lhx2 and Fezf2 in vitro induced distinct subsets of mature astrocyte genes, suggesting a modular control of maturation by different factors.
Gene expression changes induced by combined expression of Rorb and Fezf2 (Figure 6D)
Rationale The transcription factors Rorb and Fezf2 induced distinct subsets of mature astrocyte genes in vitro. To assess whether both factors directly cooperate in induce astrocyte maturation, we expressed both factors in combination in our in vitro model.
Experimental design Astrocyte were differentiated from cultured neural stem cells by growth factor withdrawal and exposure to BMP4 containing medium. At day 6 of differentiation, astrocytes were infected with lentiviral vectors to induce expression of the Rorb and Fezf2 in the same cells. RNA-Seq was performed at day 14.
Analysis outline DESEq2 was used to indentify differentially expressed genes. Gene expression was compared to immature and mature cortical astrocyte profiles and in vitro astrocytes expressing EGFP or Rorb or Fezf2 individually (data from above), focussing on the common immature/mature astrocyte-enriched genes from the cortical and striatal in vivo analyses (see above, Figures 1/2/5).
Results summary We could show that the expression of Rorb and Fezf2 in vitro induced additional mature astrocyte genes not induced by the individual factors, suggesting a cooperative synergistic activity of both factors in astrocyte maturation.
Gene expression changes induced by maturation signals (altered culture conditions) in vitro
Rationale Astrocytes in our in vitro model fail to induce many genes induced by maturation in vivo, including transcription factors like Rorb, Dbx2, Lhx2 and Fezf2, which promoted maturation in our previous experiments. We hypothesised that this might be due to a lack of extrinsic maturation signals missing in culture. We therefore assessed whether signals that have been reported to promote functional maturation (FGF2 and increased cell-cell-contacts in three-dimensional cultures), are able to induce transcription factors not induced in conventional cultures, and whether this results in improved transcriptional maturation.
Experimental design Astrocyte were differentiated from cultured neural stem cells by growth factor withdrawal and exposure to BMP4 containing medium for 7 days in 2D or 3D culture conditions and then matured in basal or FGF2 containing medium for 7 more days. RNA-Seq was performed at day 14.
Analysis outline DESEq2 was used to indentify differentially expressed genes. Gene expression was compared to immature and mature cortical astrocyte profiles (data from above), focussing on the common immature/mature astrocyte-enriched genes from the cortical and striatal in vivo analyses (see above, Figures 1/2/5).
Results summary We could show that the FGF2 and 3D culture conditions both induced mature transcription factors not induced in control cultures differentiated in basal medium in 2D. In particular, FGF2 in 3D conditions induced Rorb, Dbx2 and Lhx2 and promoted an overall more mature transcriptional profile. This suggests that indeed extrinsic maturation signals lacking in conventional cultures are required for astrocyte maturation.
Selected mature transcription factors induced by maturation signals (Figure 7B)
Selected mature genes induced by maturation signals (Figure 7C)
